Research progress in hepatitis B virus covalently closed circular DNA

Hepatitis B virus (HBV) infections are a global public health issue. HBV covalently closed circular DNA (cccDNA), the template for the transcription of viral RNAs, is a key factor in the HBV replication cycle. Notably, many host factors involved in HBV cccDNA epigenetic modulation promote the development of hepatocellular carcinoma (HCC). The HBV cccDNA minichromosome is a clinical obstacle that cannot be efficiently eliminated. In this review, we provide an update on the advances in research on HBV cccDNA and further discuss factors affecting the modulation of HBV cccDNA. Hepatitis B virus X protein (HBx) contributes to HBV cccDNA transcription and the development of hepatocarcinogenesis through modulating host epigenetic regulatory factors, thus linking the cccDNA to hepatocarcinogenesis. The measurable serological biomarkers of continued transcription of cccDNA, the effects of anti-HBV drugs on cccDNA, and potential therapeutic strategies targeting cccDNA are discussed in detail. Thus, this review describes new insights into HBV cccDNA mechanisms and therapeutic strategies for cleaning cccDNA, which will benefit patients with liver diseases.


Introduction
Chronic infection with hepatitis B virus (HBV) remains a major public health issue. Globally, more than 296 million people were chronically infected with HBV in 2019 and were therefore at risk of end-stage liver disease and hepatocellular carcinoma (HCC) [1][2][3] . HBV covalently closed circular DNA (cccDNA), the template for the transcription of viral RNAs, is a key factor in the HBV replication cycle 4,5 . In the HBV life cycle, HBV enters hepatocytes by interacting with NTCP; this is followed by uncoating and transport of the relaxed circular DNA (rcDNA) into the nucleus. As an intermediate, the cccDNA minichromosome is formed in the host cell nucleus from the rcDNA genome, which is associated with histone and non-histone proteins. The viral proteins hepatitis B core antigen (HBcAg), hepatitis B e antigen (HBeAg), HBV polymerase (pol), hepatitis B surface antigen (HBsAg), and HBV X protein (HBx) are produced from the cccDNA. The pgRNA transcribed from the cccDNA is selectively packaged inside core particles. These mature core particles can be enveloped for release as virions or transported to the nucleus to generate more cccDNA. Thus, cccDNA provides the molecular basis for establishing and maintaining viral infection [6][7][8] .
The development of HCC is affected by interactions among genetic predisposition, environmental factors, and viruses. Chronic inflammation, epigenetic modifications, DNA damage, senescence, chromosomal instability, and early neoangiogenesis drive the development and progression of HCC 9,10 . The risk of HCC is also correlated with HBV's replication, genotype, and genomic mutations [11][12][13][14] . The HBV-integrated host genome directly results in the development of HCC [15][16][17] . In addition, many other viral and host factors, such as mTOR, contribute to the development of HCC [18][19][20] . The key molecular basis of HBV persistence involves cccDNA, which plays a crucial role in the development of HCC 3 . The HBV cccDNA minichromosome is maintained throughout the clinical phases of chronic hepatitis. Because of cccDNA's key role and function in the viral replication cycle, clinical reports have indicated that measuring and eliminating cccDNA are important 6,21 .
Given the significance of cccDNA, this review discusses research progress including the composition, formation, maintenance, regulation, and epigenetic modulation of the cccDNA minichromosome; the relationships between HBxmediated cccDNA minichromosome and HCC; detection of HBV cccDNA; anti-HBV drugs targeting cccDNA; and possible curative strategies aimed at eliminating or hindering the viral cccDNA.

Mapping the HBV cccDNA minichromosome
In the nucleus, HBV cccDNA is formed from rcDNA; bound to both histones (H2A, H2B, H3, H4, and H1) and non-histone proteins (HBc, HBx, and host factors); and organized into a chromatin-like structure termed the HBV cccDNA minichromosome, which shows a typical "beads-on-a-string" arrangement under electron microscopy 22,23 . Histone proteins H2A, H2B, H3, H4, and H1 are detectable by immunoblotting on purified nucleoprotein complexes 24 . The HBV core protein (HBc) is a component of the HBV minichromosome. HBc binds the cccDNA minichromosome in vivo and in vitro, thus decreasing the nucleosomal spacing of the HBV cccDNA 24 .
Moreover, HBx in association with the cccDNA minichromosome, initiates and maintains HBV replication. HBx has been shown to activate HBV transcription through its recruitment to cccDNA and to consequently increase the recruitment of co-activators such as CBP/p300 and PCAF, which in turn target the promoters and activate gene expression, partly through histone acetylation 25-28 . Furthermore, HBx binds the parvulin 14 (Par14) and parvulin 17 (Par17) proteins, and recruits Par14/Par17 to the cccDNA minichromosome, thereby promoting the transcriptional activation of cccDNA 29 . Pre-mRNA processing factor 31 (PRPF31) is recruited to cccDNA through interacting with HBx in the nucleus, as discovered through chromatin immunoprecipitation assays 30 . Interferon-inducible protein 16 (IFI16), structural maintenance of chromosomes (SMC) complex 5/6, HP1, SMCHD1, and PML bind HBV cccDNA in hepatic nuclei, and are associated with suppression of cccDNA transcription 31-34 . APOBEC3A binds cccDNA through interacting with the HBc protein 1 . Furthermore, cellular transcription factors such as CREB, ATF, YY1, STAT1, and STAT2, and chromatin modification enzymes such as GCN5, HDAC1, SIRT1, PRMT1, PRMT5, EZH2, and SETDB1 have been shown to be associated with cccDNA through chromatin immunoprecipitation assays 25,35-37 . The lncRNA DLEU2 and HBx are co-recruited to cccDNA and subsequently play a role in the regulation of HBV 37 . Our group has reported that HAT1 confers the assembly of the cccDNA minichromosome 38 . PCNA participates in the structural organization of the cccDNA minichromosome 39 . HAT1 silencing decreases the deposition of HBx and p300 onto the cccDNA minichromosome in HBV-infected dHepaRG and HepG2-NTCP cells. HAT1 anchors to the cccDNA minichromosome through interaction with HBc, while HULC serves as a scaffold in the complex of HAT1/ HULC/HBc, thereby modulating the acetylation of histones on the cccDNA minichromosome 25,38,40 .
The mechanisms permitting the conversion from rcDNA to cccDNA remain largely unknown. Assembly of a variety of viral and host factors, including HBc, HBx, histones, and non-histone proteins, on cccDNA is key in the formation and maintenance of the cccDNA minichromosome. The HBc carboxyl-terminal domain contains the nuclear localization signal, which plays an important role in cccDNA minichromosome formation via delivering the rcDNA into the nucleus from mature nucleocapsids 41 . Mutations in the HBc N-terminal domain increase cccDNA minichromosome formation through controlling the release of rcDNA from mature capsids and the nuclear import of rcDNA 42,43 . The host ATR-CHK1 pathway is involved in cccDNA minichromosome formation through processing HBV rcDNA conversion to cccDNA 44 . In addition, SAMHD1, a component of the innate immune system that regulates the deoxyribonucleoside triphosphate levels required for host and viral DNA synthesis, has a role in regulating cccDNA minichromosome formation 45 . PRPF31 may enhance cccDNA minichromosome formation or maintenance by interacting with HBx in the nucleus 30 . Cellular DNA topoisomerase I (TOP1) and II (TOP2) are involved in catalyzing both de novo synthesis and in intracellular amplification of the cccDNA minichromosome 46 . Furthermore, several host cellular DNA repair proteins, such as tyrosyl-DNA phosphodiesterase 2 (TDP2), DNA polymerase (Pol), flap endonuclease 1 (FEN1), and DNA ligases, are required for cccDNA synthesis in de novo infection and intracellular amplification 2,47-49 . Five core components of lagging-strand synthesis have been identified and defined as the minimal set of factors essential for cccDNA minichromosome formation: proliferating cell nuclear antigen, the replication factor C complex, DNA polymerase δ, flap endonuclease 1, and DNA ligase 1 50,51 .
With respect to the roles of factors in cccDNA minichromosome maintenance, current evidence suggests that APOBEC3A and APOBEC3B anchor on the cccDNA minichromosome by interacting with HBc, thus resulting in the degradation of HBV cccDNA 1,3 . The baseline levels of APOBEC3A and APOBEC3B significantly limit the formation and accumulation of the cccDNA minichromosome 52 . High levels of ubiquitin conjugating enzyme E2 L3 (UBE2L3) maintain cccDNA stability by inducing the degradation of APOBEC3A 53 . Our group has reported that MSL2 and HULC maintain HBV cccDNA minichromosome stability through the degradation of APOBEC3B in hepatoma cells 54-56 . Furthermore, myxovirus resistance 2 (MX2) protein, an interferon-α (IFN-α) inducible effector, inhibits HBV infection by decreasing the levels of cccDNA, probably through indirectly impairing the conversion of rcDNA to cccDNA rather than destabilizing existing cccDNA 57 . The above findings are summarized in Figure 1 and Table 1.

Regulation and epigenetic modulation of the HBV cccDNA minichromosome Viral factors on the cccDNA minichromosome
Regulation of the HBV cccDNA minichromosome is mediated by viral and host factors as well as inflammatory cytokines, through epigenetic modifications of cccDNA-bound histones 5,37,58 . As a viral protein, HBx, particularly HBx amino acid residues 55-60 and 121-126 28 , play key roles in stimulating the transcription of HBV cccDNA 31 . On the one hand, HBx enables cccDNA transcription by hijacking the cellular DDB1containing E3 ubiquitin ligase, which degrades SMC5/6, a complex that binds the cccDNA minichromosome and inhibits its transcription 31 . This important mechanism provides the foundation through which HBx regulates cccDNA transcription. On the other hand, HBx prevents transcriptional repressor recruitment to the cccDNA minichromosome or recruits the transcription factors that activate the transcription of HBV genes via epigenetic regulation 25 . HBx regulates chromatin-mediated transcriptional repression of the cccDNA minichromosome through SETDB1 histone acetylation and methyl transfer, and the recruitment of heterochromatin protein 1 factor (HP1), which is correlated with condensed chromatin 32 . Similarly, HBx relieves SIRT3-mediated cccDNA transcriptional repression by inhibiting both SIRT3 expression and its recruitment to the cccDNA minichromosome 59 . In addition, HBx stimulates viral replication via DNA methylation of C-1619 in the cccDNA minichromosome 27 . Beyond viral chromatin, HBx recruits a variety of coding genes and non-coding RNA promoters associated with cccDNA, thus further regulating the cccDNA minichromosome 26,60,61 .
HBc may regulate cccDNA transcription through epigenetic modification. HBc preferentially binds CpG island 2 of the cccDNA minichromosome and alters the cccDNA minichromosome methylation profile, thus regulating the active transcription of cccDNA 62-64 . Furthermore, HBc carboxylterminal domain arginine residues in clusters III and IV may play an important role in the regulation of HBV transcription through decreasing the interaction of HBc with the cccDNA minichromosome and the acetylation of cccDNA-bound histones 65 . In addition, virion-delivered HBc stably associates with the integrated viral DNA and participates in early stages of cccDNA formation and/or transcription 66 . In contrast, a decreased amount of HBc protein on the cccDNA minichromosome does not account for the strong default of HBV RNAs in dHepaRG cells 32 .

Host factors on the cccDNA minichromosome
Beyond viral proteins, host factors also play key roles in the regulation of cccDNA transcription. IFI16 is negatively correlated with HBV and serves as a unique innate sensor that recognizes and binds the HBV cccDNA minichromosome in hepatic nuclei, thereby inhibiting cccDNA transcription and HBV replication through enhancing the recruitment of transcriptional suppressors (HDAC1, SIRT1, or EZH2) and the inhibition of transcriptional activators (p300 or CBP) anchoring to the cccDNA minichromosome 34 . However, whether HBx mediates the expression of IFI16 remains unclear.  proteasome pathway, is associated with the transcription of cccDNA. NEDD8-activating enzyme inhibitor is an efficient antiviral agent, which significantly restores SMC5/6 protein levels, and suppresses viral transcription and protein production in the HBV mini-circle system of HBV replication in both in vitro models and primary human hepatocytes infected naturally with HBV 71 . Factors such as HDM2 and ERK are associated with the activities of NEDD8 and may serve as potential targets for HBV therapy 72,73 . Host HAT1 is involved in modulating the acetylation of histones on the cccDNA minichromosome through interacting with HBc 38 . In addition, miRNAs and lncRNAs contribute to the regulation of cccDNA transcription. The lncRNA DLEU2 and HBx are co-recruited to the cccDNA minichromosome, where they displace EZH2 from the viral chromatin, and boost transcription and viral replication 37 . Another lncRNA, Hox transcript antisense intergenic RNA (HOTAIR), is associated with the transcription of cccDNA. In HBV infection, HBx induces the down-regulation of DEAD box protein 5 (DDX5), thus resulting in the transcriptional reactivation of polycomb repressive complex 2 (PRC2)/HOTAIR target genes, including HBV cccDNA-encoded genes 74 . MiR-20a may be loaded onto AGO2 before its translocation into the nucleus, thus inducing methylation of the cccDNA minichromosome in human hepatoma cells, and leading to the suppression of HBV replication 75 . MiR-548ah promotes the replication and expression of HBV through regulating its target gene HDAC4. Inhibition of HDAC4 by miRNA-548ah may influence the deacetylation state of histones binding to the cccDNA minichromosome, thus leading to the replication of cccDNA 76,77 . HBV-infected HepG2-hNTCP-C4 cells and HBV transgenic mice treated with miR-302c-3p display decreased pgRNA and HBsAg mRNA concentrations as well as amounts of cccDNA 78,79 .

HBx links the cccDNA minichro mosome to hepatocarcinogenesis
Many factors that regulate the HBV cccDNA minichromosome are involved in the development of HCC, such as HBx.
In virus-host interactions, several host factors determine the composition of the cccDNA minichromosome; host factors such as MSL2, DLEU2, HULC, and Notch signaling are regulated by HBx, thus forming a positive feedback loop 37,55,56,67,80 . Therefore, we presumed that HBx might enable HBV cccDNA transcription and the development of hepatocarcinogenesis through modulating host epigenetic regulatory factors, thereby linking cccDNA to hepatocarcinogenesis (Figure 2). HBV cccDNA and pgRNA levels represent HBV replication in the liver and might contribute to the progression of HCC in HBsAg carriers and patients with occult HBV infection 3,81,82 . Many factors may modulate cccDNA minichromosome behavior and drive hepatocarcinogenesis. The HBx-mediated biological control of the HBV cccDNA minichromosome is closely associated with the development of HBV-related HCC via host-virus interaction. HBx-activated Notch signaling may play an important role not only in HBV-related HCC but also in facilitating HBV cccDNA transcription via CREB and subsequent triggering of the downstream PKA-phospho-CREB cascade 67, 83 . Our group has reported that HBx-elevated MSL2 regulates the HBV cccDNA minichromosome in hepatoma cells, thus promoting the development of HCC and forming a positive feedback loop of HBx/MSL2/cccDNA/HBV 55 . Interestingly, antiviral therapy modulates hepatocarcinogenesis by decreasing the levels of HBx and inhibiting the tumorigenic effects of MSL2 and the cccDNA minichromosome 84 .
LncRNAs are regulators involved in biological processes, and their functional disruption has been implicated in the etiology of HCC 85 . Our group has reported that the lncRNA PCNAP1 enhances HBV replication through modulating miR-154/PCNA/HBV cccDNA signaling, in which PCNAP1/PCNA signaling drives hepatocarcinogenesis 39 . In addition, the lncRNA HULC activates HBV by modulating HBx/STAT3/miR-539/APOBEC3B signaling in HBV-related HCC. In brief, HULC enhances HBV cccDNA minichromosome stability by down-regulating APOBEC3B in hepatoma cells, thus leading to the growth of hepatoma cells by activation of HBV in vitro and in vivo 56 . Furthermore, computational modeling and biochemical evidence suggest that co-recruitment of the lncRNA DLEU2 and HBx to cccDNA displaces EZH2 from viral chromatin, thus boosting transcription and viral replication in HBV-infected cells and HBV-related HCCs 37 . The above findings are summarized in Figure 3. In this model, HBx up-regulates and recruits host factors to cccDNA, thereby enhancing the transcription of cccDNA by epigenetic regulation and leading to the replication of HBV through a positive feedback loop. Meanwhile, HBx up-regulates host factors that enhance the growth of liver cancer or directly result in the development of hepatocarcinogenesis.

Detection of HBV cccDNA
Drug development targeting cccDNA has been hindered by a lack of reliable cccDNA detection methods. Southern blot analysis is regarded as the "gold standard" for quantitative cccDNA detection and remains a widely accepted method 86 . qPCR technology is another method for cccDNA detection in laboratory settings. To date, several methods associated with qPCR have been established, such as semi-nested and nested qPCR, rolling circle amplification qPCR, and magnetic capture hybridization qPCR 87-92 . In addition, the droplet digital PCR-based cccDNA detection system is a sensitive and accurate method for quantifying cccDNA in HBV-transfected HepG2.2.15 cellular and anti-HBc-positive liver donor samples 93,94 . HBV cccDNA-selective droplet digital PCR is also sensitive in detecting cccDNA and may be a promising strategy for HBV-induced HCC surveillance and antiviral therapy evaluation 95 . Recently, a novel cccDNA quantification assay, cccDNA inversion quantitative PCR (cinqPCR), has been established, in which restriction enzymes are used to invert a DNA sequence close to the gap region of genotype D HBV strains 96 . In addition, Zhang et al. have established a highly sensitive and specific in situ hybridization assay for the detection of cccDNA in liver biopsies from patients with chronic hepatitis B (CHB), which can be used to specifically visualize the localization of cccDNA 6,97 .
However, in clinical settings, given the need for liver biopsy, detecting cccDNA in the liver of HBV-infected patients and quantifying cccDNA fluctuation during antiviral therapy through technologies such as Southern blot analysis are difficult. Currently, serum HBV DNA and HBsAg are the most widely used cccDNA markers to diagnose HBV infection and monitor antiviral therapy 98,99 . Nucleos(t)ide analogue (NA) therapy can decrease serum HBV DNA to undetectable levels, but not the levels of HBsAg and hepatitis B core-related antigen (HBcrAg), or serum HBV RNA from cccDNA. Thus, serum HBV RNA, HBsAg, and HBcrAg appear to be better surrogate markers for cccDNA than serum HBV DNA 100 . Accordingly, serum HBV RNA can serve as a biomarker to predict the natural history of disease in patients with CHB when liver biopsies are unavailable 101 . The measurement of HBV RNA before PEG-IFN based therapy has positive predictive value for maintained virological responses 102,103 . HBV RNA is a sensitive biomarker of continued transcription of cccDNA in HBeAg-negative patients, despite marked HBV DNA suppression by NAs 104 . However, no significant correlation exists between serum HBV RNA and cccDNA copy numbers 98 . Moreover, serum HBV RNA derived from pgRNA in virus-like particles is superior in reflecting the activity of intrahepatic cccDNA in patients with CHB who are receiving NA therapy 105 or in treatment-naive HBV-infected individuals [106][107][108] . Recent studies have reported that serum HBV RNA comprises heterogeneous lengths and products of incomplete reverse transcription during viral replication. Thus, the composition of HBV RNA might serve as a biomarker of cccDNA 109,110 .
Recently, a growing body of research has indicated that HBcrAg may serve as a new serum biomarker for HBV infection, treatment, and prognosis. HBcrAg contains 3 viral proteins: HBcAg, HBeAg, and a 22 kDa precore protein (p22cr) [111][112][113] . A correlation between HBcrAg levels and the size of the intrahepatic cccDNA pool has been demonstrated in cohorts of Asian genotype B/C CHB patients 114,115 . Thus, HBcrAg is useful as an HBV re-infection marker after liver transplantation and a marker in HBeAg negative CHB suppressed by NA therapy 104,116 . HBcrAg may serve as a highly sensitive marker reflecting the cccDNA content and persistence of disease even with cccDNA levels below assay detection limits 117 . Furthermore, serum HBcrAg is correlated with cccDNA transcriptional activity in CHB 118 . Nevertheless, whether HBcrAg is better than other factors as a surrogate marker for cccDNA is unclear. Serum HBcrAg is better correlated with cccDNA levels relative to HBV RNA and HBsAg in both HBeAg-positive and HBeAg-negative patients 119 . However, HBcrAg is not superior to HBV DNA and HBsAg in predicting the response during PEG-IFN treatment in white patients with HBeAg-negative CHB 120 .

IFN and cccDNA
According to cccDNA methods, the development of drug targeting cccDNA is necessary. The current standard therapy for HBV infection includes PEG-IFN-α and NAs. IFN-α may inhibit HBV viral replication through decreasing cccDNA transcription and inducing the degradation of cccDNA. Our group has reported that IFN-α epigenetically regulates the HBV cccDNA minichromosome by modulating GCN5mediated succinylation of histone H3 lysine 79 (H3K79), thereby suppressing cccDNA transcription 117 . IFN-α inhibits HBV replication by decreasing the transcription of pgRNA and subgenomic RNA through epigenetic regulation of the nuclear cccDNA minichromosome 35 . IFN-α decreases the acetylation levels of histone H3 lysine 9 (H3K9) and 27 (H3K27) in the cccDNA minichromosome, thus inducing long-lasting suppression of cccDNA transcription, and mediates a delayed response that appears to accelerate the decay of cccDNA 121 . Subsequently, the up-regulation of APOBEC3A and APOBEC3B deaminases by IFN-α and lymphotoxin-β receptor agonist have been found to cause partial degradation of cccDNA without hepatotoxicity in HBV-infected cells, primary human hepatocytes, and human liver needle biopsies 1 . An important IFN-α inducible effector, MX2, inhibits HBV infection by decreasing cccDNA minichromosome formation through conversion from rcDNA, rather than destabilizing the existing cccDNA minichromosome 57 . Moreover, UBE2L3, which is correlated with the degradation of APOBEC3A, may be involved in IFN-mediated viral suppression. IFN-α markedly inhibits the expression of UBE2L3 and consequently HBV cccDNA 53 . Three IFN-α-induced cellular proteins, STAT1, SMCHD1, and PML, may be the IFN-α response factors suppressing cccDNA transcription in the silencing of HBV replication 33 . CDM-3008, an interferon-like small chemical compound, suppresses HBV replication and decreases cccDNA levels via the activation of the JAK/STAT pathway and induction of interferon-stimulated gene (ISG) expression; the overexpression of ISG15 stimulates HBV production in an ISGylation-dependent manner 122,123 .
IFN-β, IFN-λ1, and IFN-λ2 induce the deamination and degradation of cccDNA, and are similar to IFN-α 124 . IFN-γ and TNF-α inhibit the levels of HBV cccDNA in hepatocytes through up-regulation of APOBEC3A and APOBEC3B deaminases 125 . In addition, TGF-β induces nuclear viral cccDNA degradation and hypermutation via activationinduced cytidine deaminase activity in hepatocytes 126 . Functional restoration of CD56 bright NK cells in entecavir-treated patients who are switched to PEG-IFN-α contributes to cccDNA clearance through TRAIL-induced cytolysis and TNF-α/IFNγ-mediated noncytolytic pathways 127 . IL-21based gene and cellular therapies, as valid candidates for the treatment of chronic HBV infections, have potential in removing cccDNA-bearing hepatocytes via activated CD8 + T cells together with long-term protective memory 128 . IL-6 inhibits HBV transcription by decreasing the binding of essential transcription factors such as HNF1α, HNF4α, and STAT3 to the cccDNA minichromosome, thus leading to the hypo-acetylation of cccDNA cccDNA-bound histone silencing 129 . The above findings are summarized in Figure 4.

Therapeutic strategies against cccDNA
The cccDNA minichromosome plays pivotal role in the persistence of HBV replication and therefore is a crucial target for the treatment and prognosis of HBV-related diseases, offering a possibility for HBV cure with finite therapy through affecting the assembly/formation of the cccDNA minichromosome and the transcription/stability of cccDNA.

Inhibition of assembly/formation of the cccDNA minichromosome
We presumed that blocking the assembly of the cccDNA minichromosome might be crucial for cleaning cccDNA in clinical settings. However, inhibitors of assembly of the cccDNA minichromosome have not been identified to date. HAT1 is a potential target for controlling the assembly of the cccDNA minichromosome. CCC-0975 and CCC-0346 are specific inhibitors of HBV cccDNA minichromosome formation from rcDNA 130 . HBV core protein allosteric modulators (CpAMs) inhibit the formation of nucleocapsids by disrupting the binding of pgRNA-bound polymerase and a hexamer consisting of 3 core dimers. CpAMs bind the capsid and inhibit the release of rcDNA into the nucleus 131,132 . Interestingly, CpAMs also inhibit cccDNA minichromosome formation during de novo HBV infection 133 . Moreover, hydrolyzable tannins significantly restrict cccDNA minichromosome formation and facilitate the degradation of preexisting cccDNA 134 . Therefore, agents controlling the assembly/formation of the cccDNA minichromosome provide a novel strategy for eradicating HBV cccDNA.

Decreased cccDNA transcription
Importantly, we presumed that HBx might serve as a crucial target to control cccDNA. As expected, nitazoxanide (NTZ) efficiently inhibits the HBx-DDB1 protein interaction, thus accounting for the significant restoration of SMC5/6 protein level, and suppresses viral transcription and viral protein production in the HBV minicircle system and in PHH cells naturally infected with HBV 135 . Recently, dicoumarol, an NQO1 inhibitor, has been demonstrated to have potent anti-HBV activity by promoting the degradation of HBx and blocking cccDNA transcription 136 . Spliceosome associated factor 1 (SART1) restricts the transcription of HBV cccDNA by suppressing the key HBV transcription factor HNF4α in various HBV models 137 . In addition, curcumin inhibits HBV replication through decreasing the acetylation of cccDNA-bound histones, and it may serve as a cccDNA-targeting anti-HBV agent 138 . Am80 correlates with decreased intracellular viral RNA levels, but not cccDNA copy numbers, thus indicating a persistent inhibition of HBV transcription in HepG2-NTCP cells 139 .

Disrupting cccDNA minichromosome stability
Strikingly, gene therapy targeting cccDNA is a promising technology for curing chronic HBV. Several methods, including zinc finger nucleases, transcription activator-like effector nucleases (TALENs), and the clustered regularly interspaced short palindromic repeat/CRISPR associated (CRISPR/Cas) system, have been engineered to disrupt HBV cccDNA [140][141][142][143][144][145] . The three gene editing technologies work similarly through targeting cccDNA sequences by using DNA cleaving enzymes, thus silencing cccDNA expression. However, the potential off-target effects and delivery efficiency to HBV infected hepatocytes should be addressed in the approach to eliminate cccDNA. Preclinical experiments have shown that CRISPR-Cas9-based strategies may lead to mutations and deletions that functionally inactivate cccDNA. Approximately 7% of edited DNA has been found to contain in-frame deletions, thus indicating that a single CRISPR target on the HBV genome may not inactivate cccDNA. Therefore, multiple single guide RNAs targeting different loci on the HBV genome might be required to inactivate cccDNA 131,146 . Lutgehetmann et al. 145 have evaluated the anti-HBV activity of 4 orthologous CRISPR/Cas9 systems: Streptococcus pyogenes (SpCas9), Streptococcus thermophilus (StCas9), Cas9 orthologues from Neisseria meningitidis (NmCas9), and Francisella novicida (FnCas9). Interestingly, SpCas9 and StCas9 effectively target HBV cccDNA for degradation, thereby suppressing HBV replication. StCas9 has been found to be the safest and most effective orthologous CRISPR/Cas9 for targeting HBV. The death of infected cells is a major route for the elimination of cccDNA 147 .

Indirect therapeutic strategies against cccDNA
RNA interference-based anti-HBV therapy affects cccDNA minichromosome formation via destabilizing the pgRNA and inhibiting the translation of viral proteins, such as HBx, that are important for cccDNA minichromosome formation 131,148 . The gRNA-miRNA-gRNA ternary cassette combining CRISPR/Cas9 with an RNA interference approach has shown potent activity in destroying HBV cccDNA and blocking HBV replication 144 . Furthermore, hepadnavirus-infected hepatocytes proliferation induce cccDNA dilution among daughter cells and intrahepatic cccDNA loss 145 . Moreover, NAs, as entry inhibitors, may also play roles in the regulation of cccDNA through affecting the replication of HBV 131 . HBV-specific T cells inhibit HBV replication and decrease cccDNA in infected cells; moreover, direct contact is not required for cytolysis, owing to the secretion of IFN-γ and TNF-α, thus potentially supporting HBV cure approaches 121 .
In addition, animal models are crucial in the development of anti-HBV drugs. A human chimeric liver mouse model is available to evaluate the efficacy of antiviral agents targeting the HBV replication cycle 148 and particularly to study the cccDNA in the liver. The above findings are summarized in Figure 4.

Perspectives
On the basis of mapping the cccDNA minichromosome, we provide new insights into the mechanisms through which host factors modulate the cccDNA minichromosome as well as potential treatment strategies targeting cccDNA. With the perspectives on this topic, we present suggestions for future studies on cccDNA (Figure 4), such as the many epigenetic modifying enzymes that suppress cccDNA function. Inhibitors targeting those enzymes are available to eliminate cccDNA. Combined therapy with anti-HBV drugs, such as IFN-α with inhibitors of epigenetic modification enzymes, might potentially have enhanced effectiveness in silencing or eliminating cccDNA, as well as in curing HBV-related diseases. Notably, the development of novel drugs targeting HBx will benefit the therapy of cccDNA and HBV-related cancer.

Conflict of interest statement
No potential conflicts of interest are disclosed.

Author contributions
Conceived and designed the analysis: